Quantitative phase imaging (QPI) is described in detail in Popescu, Quantitative phase imaging of cells and tissues, (McGraw-Hill, 2011), incorporated herein by reference. In QPI, the optical path length associated with substantially transparent specimens is measured and mapped, and translated into biomedically relevant information. The main figures of merit in QPI are:
1) acquisition rate;
2) transverse resolution;
3) temporal phase sensitivity; and
4) spatial phase sensitivity.
Off-axis phase imaging methods have provided the fastest acquisition rates by virtue of the fact that phase information, and thus optical path lengths, are extracted from a single recorded interferogram, as described, for example in Ikeda et al., Hilbert phase microscopy for investigating fast dynamics in transparent systems, Opt. Lett., vol. 30, pp. 1165-67 (2005), hereinafter “Ikeda (2005),” incorporated herein by reference. Diffraction-limited transverse resolution, on the other hand, is intrinsically preserved in phase shifting methods, whereas off-axis techniques may degrade transverse resolution. The highest temporal phase sensitivity (that is, the smallest frame-to-frame phase shift) is provided by common-path methods because they are the most stable. Finally, the highest spatial phase sensitivity (i.e., the smallest point-to-point phase change within the same frame) is obtained in the absence of speckles, which implies an incoherence—in at least some respect—of the source of illumination.
Diffraction phase microscopy (DPM), as described in Popescu et al., Diffraction phase microscopy for quantifying cell structure and dynamics, Opt. Lett., vol. 31, pp. 775-77 (2006), incorporated herein by reference, is both off-axis and common-path, and thus combines the benefits of fast acquisition rates and high temporal sensitivity. These features enabled DPM to perform unprecedented biological studies, especially related to red blood cell membrane dynamics. In diffraction phase microscopy, a sample is illuminated with monochromatic light such as the second harmonic of a Nd:YAG laser, for example. A phase grating is placed at the image plane (IP) of an inverted microscope where the microscope provides a magnified image of the sample. The 0th-order beam is low-pass filtered by a spatial filter in the Fourier plane of a Fourier lens, while the first order beam is allowed to pass unfiltered. The low-pass filtered (and, thus, essentially uniform) 0th-order beam and the first-order beam are recombined and interfered at a detector focal plane at a small angle with respect to the normal to the microscope output image plane, such that intensity at the detector contains a cross term dominated by the cosine of the optical phase. Further details may be found in Popescu (2006). Due to the laser illumination, however, images of DPM, as practiced, suffer from speckles, which ultimately degrade the spatial phase sensitivity and limit the applicability for studying subcellular structures.
Spatial light interference microscopy (SLIM), described, for example, in US Published Patent Application 2009/0290156 (to Popescu et al.) and by Wang et al., Spatial Light Interference Microscopy (SLIM), Opt. Exp., vol. 19, pp. 1016-26 (2011), both of which are incorporated herein by reference, removes the obstacle of speckle by using white light in a phase-shifting geometry. However, SLIM requires the acquisition of a series of four intensity images to derive a single quantitative phase image.
A subsequent quantitative phase imaging modality, introduced by Ding et al., Instantaneous spatial light interference microscopy, Optics Exp., vol. 18, pp. 1569-75 (2010), incorporated herein by reference, places a phase contrast condenser annulus in the illuminating beam and a diffraction grating at the image plane (IP) of an inverted microscope, generating distinct diffraction orders. The 0th order is spatially low-pass filtered to match the image of the condenser annulus, passing the DC component of the 0th order to serve as a reference beam which is interfered with the first order, without the spatial offset that characterizes DPM, in order to derive a spectrally resolved phase image. iSLIM requires a condenser annulus at the expense of illumination available at higher spatial frequencies of the diffracted component, thereby reducing the signal-to-noise in the phase image. Furthermore, the iSLIM system requires a spatial light modulator (KM) for filtering the unscattered light which makes the iSLIM setup more complicated.
It would thus be desirable to obtain the benefit of speckle-free quantitative phase imaging, but to do so in a single exposure.